Or low conc of abeta is the issue???running tricine gel will say something?

I don't fully understand the details of your description, but it sounds to me that your Abeta aggregates may be settling out of solution. Hence, you get a better fluorescence signal, if you stir the solution and bring the Abeta aggregates back into the liquid phase.

Do you see any particles / aggregates by eye? 

Dear Holger

let me explain again. I prepared aggregating samples of abeta 40 (incubated for 2-3 days at 37 with continuous agitation). Then on the day of ThT assay, i prepared gly NaOH buffer and tht dye solution. I took an eppendrof : added 30ul of agg abeta, 30ul of ThT (100mM stock) and 240 ul of glycine buffer (500mM stock) and mixed and vortexed it. Then syringed into quartz 0.5ml cuvette and scanned in cary varian fluorescence spectrometer. I am scanning with and without stirrer. without stir, i dont see much signal but intensity increases when i dip in the stir into the cuvette. The problem is that when i shift the cuvetter or  redip the sirrer into the cuvette at any other point of time signal changes (i did not add any further samples into the cuvette). For some of the samples, I have seen disc shaped aggregate particles (whitish color), could these be fibrillar aggregates?

When you dilute your sample in the glycine buffer at pH 8.5, what made you choose that pH? I suspect that such a high pH value might (partially) denature your sample. The ThT assay certainly doesn't need that pH and works in a wide variety of buffer conditions.

This protocol is a nature protocol 2010 that I followed. The reason for that high pH it seems that at high pH ThT has high fluorescence signal. What I understand why ppl use this buffer may be for locking the type of aggregate you are looking after aggregation protocol. Am i correct? this is what I read? I think I can still the same buffer i used to aggregate samples (sodium phosphate buffer). The thing is i tried to immitate the protocol so did not alter anything to achieve  data.

In the experiments I was involved in we kept the pH the same as the fibrillization conditions and obtained good ThT fluorescence signals. Maybe they would have been higher at a higher pH, but we didn't disrupt or change the aggregation state to any noticeable degree.

Maybe it'll be worthwhile to try your ThT assay in the same buffer as your fibrillization reaction.

thanks prof holger for reply. could you please let me know when the aggregates are visible by eys what are the features? do they have a shape or thread like whitish material? how would i be sure if I can measure them on TEM? or how would i know specifically  that prefibrillar aggregates or oligomers are present in samples which would show different TEM image?

Hi Anup,

Whenever you see particles in your solution by eye, they are very large (at least to an electron microscopist). If they are thin and whispy looking they are often fibrillar in nature, otherwise they could also be amorphous aggregates. Sometimes large aggregates are difficult to visualize with negative stain EM, since you can wash them off the grid easily during the staining process.

Prefibrillar aggregates and oligomers would be completely invisible to the naked eye due to their small size, i.e. the solution would be perfectly clear. But these aggregate sizes are much easier to visualize with negative stain EM, since they are not easily washed off the grid.

Hi Anup,

I don't know as much about a-beta, but pretty sure I read they also respond to hydrophobic interfaces. I did a paper on how PTFE (teflon) is sufficient to initiate amyloid formation  from otherwise stable monomers of alpha-synuclein.

Your stir bar is most likely coated with... PTFE.

Here's the paper. Avoid air bubbles too if you want to be clean about your experiment. http://www.ncbi.nlm.nih.gov/pubmed/20578692

Jeremy

Thanks prof holger and Jeremy 

Hi Anup,

If the fibrillation is "too" productive, you may get amyloid fibrils that bind the thioflavin T with smaller affinity.  Thus, you will get less fluorescence activity.  When I was doing amyloid research, I would give the fibril solution a good vortex on a regular table-top vortexer for two minutes before sampling with ThT.  In my experience, protofibrils, which are prefibrils or broken fibrils, fluoresce better than mature fibrils.  By vortexing you break up the mature fibrils into shorter protofibrils.  pH 8.5 should not be an issue but you are right about high ThT background.